S1PR3 agonism and S1P lyase inhibition rescue mice in the severe state of experimental sepsis.

Sepsis is characterized as life-threatening organ dysfunction caused by a dysregulated host response to an infection. Despite numerous clinical trials that addressed this syndrome, there is still no causative treatment available to dampen its severity. Curtailing the infection at an early stage with anti-infectives is the only effective treatment regime besides intensive care. In search for additional treatment options, we recently discovered the inhibition of the sphingosine 1-phosphate (S1P) lyase and subsequent activation of the S1P receptor type 3 (S1PR3) in pre-conditioning experiments as promising targets for sepsis prevention. Here, we demonstrate that treatment of septic mice with the direct S1P lyase inhibitor C31 or the S1PR3 agonist CYM5541 in the advanced phase of sepsis resulted in a significantly increased survival rate. A single dose of each compound led to a rapid decline of sepsis severity in treated mice and coincided with decreased cytokine release and increased lung barrier function with unaltered bacterial load. The survival benefit of both compounds was completely lost in S1PR3 deficient mice. Treatment of the murine macrophage cell line J774.1 with either C31 or CYM5541 resulted in decreased protein kinase B (Akt) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) phosphorylation without alteration of the mitogen-activated protein kinase (MAPK) p38 and p44/42 phosphorylation. Thus, activation of S1PR3 in the acute phase of sepsis by direct agonism or S1P lyase inhibition dampened Akt and JNK phosphorylation, resulting in decreased cytokine release, improved lung barrier stability, rapid decline of sepsis severity and better survival in mice.

Sphingosine-1-phosphate suppresses GLUT activity through PP2A and counteracts hyperglycemia in diabetic red blood cells.

Red blood cells (RBC) are the major carriers of sphingosine-1-phosphate (S1P) in blood. Here we show that variations in RBC S1P content achieved by altering S1P synthesis and transport by genetic and pharmacological means regulate glucose uptake and metabolic flux. This is due to S1P-mediated activation of the catalytic protein phosphatase 2 (PP2A) subunit leading to reduction of cell-surface glucose transporters (GLUTs). The mechanism dynamically responds to metabolic cues from the environment by increasing S1P synthesis, enhancing PP2A activity, reducing GLUT phosphorylation and localization, and diminishing glucose uptake in RBC from diabetic mice and humans. Functionally, it protects RBC against lipid peroxidation in hyperglycemia and diabetes by activating the pentose phosphate pathway. Proof of concept is provided by the resistance of mice lacking the S1P exporter MFSD2B to diabetes-induced HbA1c elevation and thiobarbituric acid reactive substances (TBARS) generation in diabetic RBC. This mechanism responds to pharmacological S1P analogues such as fingolimod and may be functional in other insulin-independent tissues making it a promising therapeutic target.

Immunometabolic capacities of nutritional fatty acids in regulation of inflammatory bone cell interaction and systemic impact of periodontal infection.

Introduction: Novel preventive strategies in periodontal disease target the bacterial-induced inflammatory host response to reduce associated tissue destruction. Strategies focus on the modulation of tissue-destroying inflammatory host response, particularly the reduction of inflammation and promotion of resolution. Thereby, nutrition is a potent immunometabolic non-pharmacological intervention. Human studies have demonstrated the benefit of olive oil-containing Mediterranean-style diets (MDs), the main component of which being mono-unsaturated fatty acid (FA) oleic acid (OA (C18:1)). Hence, nutritional OA strengthened the microarchitecture of alveolar trabecular bone and increased circulating pro-resolving lipid mediators following bacterial inoculation with periodontal pathogen Porphyromonas gingivalis, contrary to saturated FA palmitic acid (PA (C16:0)), which is abundant in Western-style diets. Additionally, the generalized distribution of inflammatory pathway mediators can occur in response to bacterial infection and compromise systemic tissue metabolism and bone homeostasis distant from the side of infection. Whether specific FA-enriched nutrition and periodontal inoculation are factors in systemic pathology that can be immune-modulatory targeted through dietary substitution is unknown and of clinical relevance. Methods: Normal-weight C57BL/6-mice received OA-or PA-enriched diets (PA-ED, OA-ED, PA/OA-ED) or a normal-standard diet (n=12/group) for 16 weeks and were orally infected with P. gingivalis/placebo to induce periodontal disease. Using histomorphometry and LC-MS/MS, systemic bone morphology, incorporated immunometabolic FA-species, serological markers of bone metabolism, and stress response were determined in addition to bone cell inflammation and interaction in vitro. Results: In contrast to OA-ED, PA-ED reduced systemic bone microarchitecture paralleled by increased lipotoxic PA-containing metabolite accumulation in bone. Substitution with OA reversed the bone-destructive impact of PA, which was accompanied by reduced diacylglycerols (DAG) and saturated ceramide levels. Further, PA-associated reduction in mineralization activity and concomitant pro-inflammatory activation of primary osteoblasts were diminished in cultures where PA was replaced with OA, which impacted cellular interaction with osteoclasts. Additionally, PA-ED increased osteoclast numbers in femurs in response to oral P. gingivalis infection, whereas OA-ED reduced osteoclast occurrence, which was paralleled by serologically increased levels of the stress-reducing lipokine PI(18:1/18:1). Conclusion: OA substitution reverses the bone-destructive and pro-inflammatory effects of PA and eliminates incorporated lipotoxic PA metabolites. This supports Mediterranean-style OA-based diets as a preventive intervention to target the accumulation of PA-associated lipotoxic metabolites and thereby supports systemic bone tissue resilience after oral bacterial P. gingivalis infection.

Revealing concealed cardioprotection by platelet Mfsd2b-released S1P in human and murine myocardial infarction.

Antiplatelet medication is standard of care in acute myocardial infarction (AMI). However, it may have obscured beneficial properties of the activated platelet secretome. We identify platelets as major source of a sphingosine-1-phosphate (S1P) burst during AMI, and find its magnitude to favorably associate with cardiovascular mortality and infarct size in STEMI patients over 12 months. Experimentally, administration of supernatant from activated platelets reduces infarct size in murine AMI, which is blunted in platelets deficient for S1P export (Mfsd2b) or production (Sphk1) and in mice deficient for cardiomyocyte S1P receptor 1 (S1P1). Our study reveals an exploitable therapeutic window in antiplatelet therapy in AMI as the GPIIb/IIIa antagonist tirofiban preserves S1P release and cardioprotection, whereas the P2Y12 antagonist cangrelor does not. Here, we report that platelet-mediated intrinsic cardioprotection is an exciting therapeutic paradigm reaching beyond AMI, the benefits of which may need to be considered in all antiplatelet therapies.

Glucocorticoid activation of anti-inflammatory macrophages protects against insulin resistance.

Insulin resistance (IR) during obesity is linked to adipose tissue macrophage (ATM)-driven inflammation of adipose tissue. Whether anti-inflammatory glucocorticoids (GCs) at physiological levels modulate IR is unclear. Here, we report that deletion of the GC receptor (GR) in myeloid cells, including macrophages in mice, aggravates obesity-related IR by enhancing adipose tissue inflammation due to decreased anti-inflammatory ATM leading to exaggerated adipose tissue lipolysis and severe hepatic steatosis. In contrast, GR deletion in Kupffer cells alone does not alter IR. Co-culture experiments show that the absence of GR in macrophages directly causes reduced phospho-AKT and glucose uptake in adipocytes, suggesting an important function of GR in ATM. GR-deficient macrophages are refractory to alternative ATM-inducing IL-4 signaling, due to reduced STAT6 chromatin loading and diminished anti-inflammatory enhancer activation. We demonstrate that GR has an important function in macrophages during obesity by limiting adipose tissue inflammation and lipolysis to promote insulin sensitivity.

Sphingosine-1-phosphate improves outcome of no-reflow acute myocardial infarction via sphingosine-1-phosphate receptor 1.

AIMS: Therapeutic options targeting post-ischaemic cardiac remodelling are sparse. The bioactive sphingolipid sphingosine-1-phosphate (S1P) reduces ischaemia/reperfusion injury. However, its impact on post-ischaemic remodelling independently of its infarct size (IS)-reducing effect is yet unknown and was addressed in this study. METHODS AND RESULTS: Acute myocardial infarction (AMI) in mice was induced by permanent ligation of the left anterior descending artery (LAD). C57Bl6 were treated with the S1P lyase inhibitor 4-deoxypyridoxine (DOP) starting 7 days prior to AMI to increase endogenous S1P concentrations. Cardiac function and myocardial healing were assessed by cardiovascular magnetic resonance imaging (cMRI), murine echocardiography, histomorphology, and gene expression analysis. DOP effects were investigated in cardiomyocyte-specific S1P receptor 1 deficient (S1PR1 Cardio Cre+) and Cre- control mice and S1P concentrations measured by LC-MS/MS. IS and cardiac function did not differ between control and DOP-treated groups on day one after LAD-ligation despite fourfold increase in plasma S1P. In contrast, cardiac function was clearly improved and myocardial scar size reduced, respectively, on Day 21 in DOP-treated mice. The latter also exhibited smaller cardiomyocyte size and reduced embryonic gene expression. The benefit of DOP treatment was abolished in S1PR1 Cardio Cre+. CONCLUSIONS: S1P improves cardiac function and myocardial healing post AMI independently of initial infarct size and accomplishes this via the cardiomyocyte S1PR1. Hence, in addition to its beneficial effects on I/R injury, S1PR1 may be a promising target in post-infarction myocardial remodelling as adjunctive therapy to revascularization as well as in patients not eligible for standard interventional procedures.

The Impact of Sphingosine Kinases on Inflammation-Induced Cytokine Release and Vascular Endothelial Barrier Integrity.

Sphingosine kinases type 1 and 2 (SphK1/2) are required for the production of the immune modulator sphingosine 1-phosphate (S1P). SphK1 deficient mice (SphK1-/-) revealed 50% reduced S1P in plasma, while SphK2-/- mice demonstrated 2-3 times increased S1P levels in plasma. Since plasma S1P is a potent inducer of vascular endothelial cell (VEC) barrier stability, we hypothesized that higher and lower levels of S1P in SphK2-/- and SphK1-/- mice, respectively, compared to wild type (wt) mice should translate into decreased and increased severity of induced systemic inflammation due to improved or damaged VEC barrier maintenance. To our surprise, both SphK1-/- and SphK2-/- mice showed improved survival rate and earlier recovery from inflammation-induced weight loss compared to wt mice. While no difference was observed in VEC barrier stability by monitoring Evans blue leakage into peripheral tissues, SphK1-/- mice demonstrated a distinct delay and SphK2-/- mice an improved resolution of early pro-inflammatory cytokine release in plasma. Ex vivo cell culture experiments demonstrated that bone marrow-derived dendritic cells (BMDC) generated from SphK1-/- and SphK2-/- mice responded with decreased interferon-γ (IFN-γ) production upon stimulation with lipopolysaccharides (LPS) compared to wt BMDC, while activation-induced cytokine expression of lymphocytes and macrophages was not majorly altered. Ex vivo stimulation of macrophages with IFN-γ resulted in increased cytokine release. These results suggest that SphK1/2 are involved in production and secretion of IFN-γ by DC. DC-derived IFN-γ subsequently stimulates the production and secretion of a large panel of inflammatory cytokines by macrophages, which belong to the main cytokine-releasing cells of the early innate immune response. Inhibitors of SphK1/2 may therefore be attractive targets to dampen the early cytokine response of macrophages as part of the innate immune response.

Sphingosine-1-Phosphate (S1P) Lyase Inhibition Aggravates Atherosclerosis and Induces Plaque Rupture in ApoE-/-Mice.

Altered plasma sphingosine-1-phosphate (S1P) concentrations are associated with clinical manifestations of atherosclerosis. However, whether long-term elevation of endogenous S1P is pro- or anti-atherogenic remains unclear. Here, we addressed the impact of permanently high S1P levels on atherosclerosis in cholesterol-fed apolipoprotein E-deficient (ApoE-/-) mice over 12 weeks. This was achieved by pharmacological inhibition of the S1P-degrading enzyme S1P lyase with 4-deoxypyridoxine (DOP). DOP treatment dramatically accelerated atherosclerosis development, propagated predominantly unstable plaque phenotypes, and resulted in frequent plaque rupture with atherothrombosis. Macrophages from S1P lyase-inhibited or genetically deficient mice had a defect in cholesterol efflux to apolipoprotein A-I that was accompanied by profoundly downregulated cholesterol transporters ATP-binding cassette transporters ABCA1 and ABCG1. This was dependent on S1P signaling through S1PR3 and resulted in dramatically enhanced atherosclerosis in ApoE-/-/S1PR3-/- mice, where DOP treatment had no additional effect. Thus, high endogenous S1P levels promote atherosclerosis, compromise cholesterol efflux, and cause genuine plaque rupture.

Altered Serum Phospholipids in Atopic Dermatitis and Association with Clinical Status.

Circulating phospholipids have been considered as biomarkers and therapeutic targets in multiple disorders. Atopic dermatitis (AD) is the most common inflammatory skin disease. Although there are numerous studies having addressed stratum corneum lipids in the context of epidermal barrier, little is known about the circulating lipids in patients with AD. In this study, we explored the changes of serum phospholipids in AD using liquid chromatography coupled to tandem mass spectrometry and sought serum lipids' contribution to clinical status. Several serum levels of phospholipids were altered in the AD group (n = 179) compared with that in healthy controls (n = 47) and patients without AD with atopic comorbidities (n = 22); lipids exhibiting the apparent changes included increased sphingosine, multiple variants of phosphatidylcholine, and decreased ceramide (16:0) in patients with AD. Moreover, serum levels of sphingosine correlated with the severity of AD, and sphingosine and ceramide(16:0) were also detected as the risk-increasing effect and risk-reduction effect of AD, respectively. In summary, alterations in the serum concentration of phospholipids are seen in patients with AD. Although more detailed investigations will be needed to evaluate the significance of the changes in circulating lipids in AD, these findings can provide, to our knowledge, previously unreported insight into AD's pathogenesis and therapeutic strategies.

Erythrocytes increase endogenous sphingosine 1-phosphate levels as an adaptive response to SARS-CoV-2 infection.

Low plasma levels of the signaling lipid metabolite sphingosine 1-phosphate (S1P) are associated with disrupted endothelial cell (EC) barriers, lymphopenia and reduced responsivity to hypoxia. Total S1P levels were also reduced in 23 critically ill patients with coronavirus disease 2019 (COVID-19), and the two main S1P carriers, serum albumin (SA) and high-density lipoprotein (HDL) were dramatically low. Surprisingly, we observed a carrier-changing shift from SA to HDL, which probably prevented an even further drop in S1P levels. Furthermore, intracellular S1P levels in red blood cells (RBCs) were significantly increased in COVID-19 patients compared with healthy controls due to up-regulation of S1P producing sphingosine kinase 1 and down-regulation of S1P degrading lyase expression. Cell culture experiments supported increased sphingosine kinase activity and unchanged S1P release from RBC stores of COVID-19 patients. These observations suggest adaptive mechanisms for maintenance of the vasculature and immunity as well as prevention of tissue hypoxia in COVID-19 patients.

Intracellularly Released Cholesterol from Polymer-Based Delivery Systems Alters Cellular Responses to Pneumolysin and Promotes Cell Survival.

Cholesterol is highly abundant within all human body cells and modulates critical cellular functions related to cellular plasticity, metabolism, and survival. The cholesterol-binding toxin pneumolysin represents an essential virulence factor of Streptococcus pneumoniae in establishing pneumonia and other pneumococcal infections. Thus, cholesterol scavenging of pneumolysin is a promising strategy to reduce S. pneumoniae induced lung damage. There may also be a second cholesterol-dependent mechanism whereby pneumococcal infection and the presence of pneumolysin increase hepatic sterol biosynthesis. Here we investigated a library of polymer particles varying in size and composition that allow for the cellular delivery of cholesterol and their effects on cell survival mechanisms following pneumolysin exposure. Intracellular delivery of cholesterol by nanocarriers composed of Eudragit E100-PLGA rescued pneumolysin-induced alterations of lipid homeostasis and enhanced cell survival irrespective of neutralization of pneumolysin.

Long-Chain and Very Long-Chain Ceramides Mediate Doxorubicin-Induced Toxicity and Fibrosis.

Doxorubicin (Dox) is a chemotherapeutic agent with cardiotoxicity associated with profibrotic effects. Dox increases ceramide levels with pro-inflammatory effects, cell death, and fibrosis. The purpose of our study was to identify the underlying ceramide signaling pathways. We aimed to characterize the downstream effects on cell survival, metabolism, and fibrosis. Human fibroblasts (hFSF) were treated with 0.7 µM of Dox or transgenically overexpressed ceramide synthase 2 (FLAG-CerS2). Furthermore, cells were pre-treated with MitoTempo (MT) (2 h, 20 µM) or Fumonisin B1 (FuB) (4 h, 100 µM). Protein expression was measured by Western blot or immunofluorescence (IF). Ceramide levels were determined with mass spectroscopy (MS). Visualizations were conducted using laser scanning microscopy (LSM) or electron microscopy. Mitochondrial activity was measured using seahorse analysis. Dox and CerS2 overexpression increased CerS2 protein expression. Coherently, ceramides were elevated with the highest peak for C24:0. Ceramide- induced mitochondrial ROS production was reduced with MT or FuB preincubation. Mitochondrial homeostasis was reduced and accompanied by reduced ATP production. Our data show that the increase in pro-inflammatory ceramides is an essential contributor to Dox side-effects. The accumulation of ceramides resulted in a lipotoxic shift and subsequently mitochondrial structural and functional damage, which was partially reversible following inhibition of ceramide synthesis.

Sphingosine-1 Phosphate Lyase Regulates Sensitivity of Pancreatic Beta-Cells to Lipotoxicity.

Elevated levels of free fatty acids (FFAs) have been related to pancreatic beta-cell failure in type 2 diabetes (T2DM), though the underlying mechanisms are not yet fully understood. FFAs have been shown to dysregulate formation of bioactive sphingolipids, such as ceramides and sphingosine-1 phosphate (S1P) in beta-cells. The aim of this study was to analyze the role of sphingosine-1 phosphate lyase (SPL), a key enzyme of the sphingolipid pathway that catalyzes an irreversible degradation of S1P, in the sensitivity of beta-cells to lipotoxicity. To validate the role of SPL in lipotoxicity, we modulated SPL expression in rat INS1E cells and in human EndoC-ßH1 beta-cells. SPL overexpression in INS1E cells (INS1E-SPL), which are characterized by a moderate basal expression level of SPL, resulted in an acceleration of palmitate-mediated cell viability loss, proliferation inhibition and induction of oxidative stress. SPL overexpression affected the mRNA expression of ER stress markers and mitochondrial chaperones. In contrast to control cells, in INS1E-SPL cells no protective effect of oleate was detected. Moreover, Plin2 expression and lipid droplet formation were strongly reduced in OA-treated INS1E-SPL cells. Silencing of SPL in human EndoC-ßH1 beta-cells, which are characterized by a significantly higher SPL expression as compared to rodent beta-cells, resulted in prevention of FFA-mediated caspase-3/7 activation. Our findings indicate that an adequate control of S1P degradation by SPL might be crucially involved in the susceptibility of pancreatic beta-cells to lipotoxicity.

Targeted delivery of a phosphoinositide 3-kinase γ inhibitor to restore organ function in sepsis.

Jaundice, the clinical hallmark of infection-associated liver dysfunction, reflects altered membrane organization of the canalicular pole of hepatocytes and portends poor outcomes. Mice lacking phosphoinositide 3-kinase-γ (PI3Kγ) are protected against membrane disintegration and hepatic excretory dysfunction. However, they exhibit a severe immune defect that hinders neutrophil recruitment to sites of infection. To exploit the therapeutic potential of PI3Kγ inhibition in sepsis, a targeted approach to deliver drugs to hepatic parenchymal cells without compromising other cells, in particular immune cells, seems warranted. Here, we demonstrate that nanocarriers functionalized through DY-635, a fluorescent polymethine dye, and a ligand of organic anion transporters can selectively deliver therapeutics to hepatic parenchymal cells. Applying this strategy to a murine model of sepsis, we observed the PI3Kγ-dependent restoration of biliary canalicular architecture, maintained excretory liver function, and improved survival without impairing host defense mechanisms. This strategy carries the potential to expand targeted nanomedicines to disease entities with systemic inflammation and concomitantly impaired barrier functionality.

Barrier maintenance by S1P during inflammation and sepsis.

Sphingosine 1-phosphate (S1P) is a multifaceted lipid signaling molecule that activates five specific G protein-coupled S1P receptors. Despite the fact that S1P is known as one of the strongest barrier-enhancing molecules for two decades, no medical application is available yet. The reason for this lack of translation into clinical practice may be the complex regulatory network of S1P signaling, metabolism and transportation.In this review, we will provide an overview about the physiology and the network of S1P signaling with the focus on endothelial barrier maintenance in inflammation. We briefly describe the physiological role of S1P and the underlying S1P signaling in barrier maintenance, outline differences of S1P signaling and metabolism in inflammatory diseases, discuss potential targets and compounds for medical intervention, and summarize our current knowledge regarding the role of S1P in the maintenance of specialized barriers like the blood-brain barrier and the placenta.

Activation of Sphingomyelinase-Ceramide-Pathway in COVID-19 Purposes Its Inhibition for Therapeutic Strategies.

Effective treatment strategies for severe coronavirus disease (COVID-19) remain scarce. Hydrolysis of membrane-embedded, inert sphingomyelin by stress responsive sphingomyelinases is a hallmark of adaptive responses and cellular repair. As demonstrated in experimental and observational clinical studies, the transient and stress-triggered release of a sphingomyelinase, SMPD1, into circulation and subsequent ceramide generation provides a promising target for FDA-approved drugs. Here, we report the activation of sphingomyelinase-ceramide pathway in 23 intensive care patients with severe COVID-19. We observed an increase of circulating activity of sphingomyelinase with subsequent derangement of sphingolipids in serum lipoproteins and from red blood cells (RBC). Consistent with increased ceramide levels derived from the inert membrane constituent sphingomyelin, increased activity of acid sphingomyelinase (ASM) accurately distinguished the patient cohort undergoing intensive care from healthy controls. Positive correlational analyses with biomarkers of severe clinical phenotype support the concept of an essential pathophysiological role of ASM in the course of SARS-CoV-2 infection as well as of a promising role for functional inhibition with anti-inflammatory agents in SARS-CoV-2 infection as also proposed in independent observational studies. We conclude that large-sized multicenter, interventional trials are now needed to evaluate the potential benefit of functional inhibition of this sphingomyelinase in critically ill patients with COVID-19.

Development and validation of a QTrap method for sensitive quantification of sphingosine 1-phosphate.

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid and ligand for five G protein-coupled cell-surface receptors designated S1PR1-5. The determination of low levels of S1P remains a challenge and usually requires sophisticated analytical instrumentation and methodology. This report describes a technique using the linear ion trap mode of a basic QTrap triple-quadrupole mass spectrometer. S1P was extracted from acidified biological samples using a modified Folch extraction procedure. After the addition of C17-sphingosine as an internal standard, a step gradient LC method was used to separate the analytes on a reversed-phase C18 MultoHigh analytical column. After the internal standard C17-sphingosine was detected by multiple reaction monitoring (MRM), the detection mode was switched to enhanced product ion (EPI) mode for the detection of S1P. The mode was switched back to MRM again for the detection of other analytes. Using this QTrap method, we reached a limit of detection of 1 nM and a limit of quantification of 3 nM for S1P, which was up to 30 times more sensitive than the MRM mode with the same instrument. Intra-day precision ranged between -3.8 and 6.3%, and inter-day precision was between -13.8 and 3.3%, depending on the spiked S1P concentration.

Sphingosine 1-phosphate in sepsis and beyond: Its role in disease tolerance and host defense and the impact of carrier molecules.

Sphingosine 1-phosphate (S1P) is an important immune modulator responsible for physiological cellular responses like lymphocyte development and function, positioning and emigration of T and B cells and cytokine secretion. Recent reports indicate that S1P does not only regulate immunity, but can also protect the function of organs by inducing disease tolerance. S1P also influences the replication of certain pathogens, and sphingolipids are also involved in pathogen recognition and killing. Certain carrier molecules for S1P like serum albumin and high density lipoproteins contribute to the regulation of S1P effects. They are able to associate with S1P and modulate its signaling properties. Similar to S1P, both carrier molecules are also decreased in sepsis patients and likely contribute to sepsis pathology and severity. In this review, we will introduce the concept of disease tolerance and the involvement of S1P. We will also discuss the contribution of S1P and its precursor sphingosine to host defense mechanisms against pathogens. Finally, we will summarize current data demonstrating the influence of carrier molecules for differential S1P signaling. The presented data may lead to new strategies for the prevention and containment of sepsis.

Validation of a monoclonal antibody directed against the human sphingosine 1-phosphate receptor type 1.

The sphingosine 1-phosphate receptor type 1 (S1PR1) has several important functions, including stabilizing endothelial barrier and maintaining lymphocyte circulation. These functions are critically dependent on the regulation of S1PR1 cell surface expression. Currently available antibodies against human S1PR1 are not able to pick up cell surface expression on living cells by flow cytometry due to intracellular epitopes or unspecific binding. Here we describe the generation of a mouse monoclonal antibody specific for the N-terminal region of human S1PR1. It has an immunoglobulin M (IgM) kappa isotype and detects cell surface expression of recombinant human S1PR1 on overexpressing cells. Due to unspecific intracellular cell staining, it cannot be used for staining of dead cells and tissue slides or in microscopic analyses. It is also not suitable for Western blot analysis and immunoprecipitation. However, the antibody can stain for endogenous S1PR1 on human endothelial cell lines and primary human umbilical vein endothelial cells (HUVEC). Incubation of these cells with various S1PR1 agonists revealed potent S1PR1 internalization, which was not the case with the specific antagonist W146. Surprisingly, human T and B cells isolated from blood and palatine tonsils did not show specific staining, demonstrating significantly lower endogenous S1PR1 surface expression on lymphocytes than on endothelial cells.